Circadian rhythms are generated by molecular clocks that are universally used to synchronize behavior and physiology with the 24-hour solar cycle. This proposal seeks to understand the biochemical basis of circadian timing and photoentrainment, the process by which molecular clocks are aligned to the external light/dark cycle. In mammals, circadian rhythms arise from set of interlocked transcription feedback loops involving dedicated clock proteins: at the center of this network, CLOCK:BMAL1 activates transcription of its repressors PERIOD (PER) and CRYPTOCHROME (CRY), which ultimately feed back to complete a ~24-hour long cycle of gene activation and repression. Photoentrainment is important to keep molecular clocks on track with environmental light cycles. Chronic circadian misalignment (i.e. jetlag) leads to increased risk for metabolic disorders, cardiovascular disease and cancer due to disruption of the systemic control of physiology by circadian rhythms. While much of the photoentrainment pathway has been laid out from ocular photoreception to its acute induction of Per mRNA in the master clock of the brain, crucially, the final biochemical steps that execute entrainment on the molecular level remain completely unknown. Exposure to light before dawn leads to phase advances of the molecular clock, while light after dusk delays the clock, both of which keep CLOCK:BMAL1 activity aligned with the day. How does this plasticity in phase shifting arise from the same light-dependent induction of Per mRNA that occurs at dusk and dawn? ChIP-seq studies provide evidence for distinct repressive complexes that assemble in the evening, an `early' complex of PER and CRY proteins that assembles at dusk on CLOCK:BMAL1 and a `late' complex of CRY1 bound alone to CLOCK:BMAL1 at dawn. Our central hypothesis is that differences in the composition of early and late repressive complexes are exploited for entrainment, leading to differential regulation by light-induced PER2 at dusk and dawn. We will test this with three specific aims. First, the molecular basis for differences in regulation of CLOCK:BMAL1 by CRY1, CRY2 and PER2 will be defined with biochemical, biophysical, and cellular studies. Second, structures of early and late repressive complexes will be determined by cryo-electron microscopy to identify overall changes in molecular architecture of the core clock proteins that occur throughout the evening. Third, the development of a new optogenetic model for the study of cellular clocks will allow the identification of biochemical determinants by which clocks are entrained to external stimuli. Collectively, these lines of study will address how core clock proteins interact throughout the evening in distinct regulatory complexes to generate circadian timekeeping and respond to external stimuli.